Biodegradable polyacetals for in vivo polynucleotide delivery

ABSTRACT

Degradable complexes comprising a polycation, a polyanion and a polynucleotide are useful for in vivo polynucleotide delivery applications.

RELATED APPLICATION INFORMATION

This application is a divisional of U.S. application Ser. No.10/946,383, filed Sep. 21, 2004, now U.S. Pat. No. 7,794,696, whichclaims the benefit of U.S. Provisional Patent Application No.60/507,161, filed Sep. 29, 2003, which is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to degradable polymer compositionsuseful for in vitro and in vivo polynucleotide delivery applications.More particularly, this invention relates to degradable complexescomprising polyanions, polycations and polynucleotides useful for invivo polynucleotide delivery applications in mammals.

2. Description of the Related Art

There is a need for non-viral drug delivery systems having desirableproperties such as low immunogenicity, amenable to production on arelatively large scale, and which can be easily modified to provide arange of biological properties. See Mulligan, R. C., “The basic scienceof gene therapy,” Science 260, 926-932 (1993); and Luo, D. & Saltzman,W. M. “Synthetic DNA delivery systems,” Nat. Biotechnol. 18, 33-37(2000). However, non-degradable cationic polymers such as poly(lysine)and polyethyleneimine (PEI) can have significant cytotoxicity. SeeChoksakulnimitr, S., Masuda, S., Tokuda, H., Takakura, Y. & Hashida, M.,“In vitro cytotoxicity of macromolecules in different cell culturesystems,” J. Control Release 34, 233-241 (1995); Brazeau, G. A., Attia,S., Poxon, S. & Hughes, J. A., “In Vitro Myotoxicity of Selectedcationic macrolecules used in non-viral gene therapy,” Pharm. Res. 15,680-684 (1998); and Ahn, C.-H., Chae, S. Y., Bae, Y. H. & Kim, S. W.“Biodegradable poly(ethylenimine) for plasmid DNA delivery,” J. Control.Release 80, 273-282 (2002).

To reduce cytotoxicity, some efforts have been made to developdegradable cationic polymers (polycations). See Ahn, C.-H., Chae, S. Y.,Bae, Y. H. & Kim, S. W., “Biodegradable poly(ethylenimine) for plasmidDNA delivery,” J. Control. Release 80, 273-282 (2002); Lynn, D. M.;Anderson, D. G.; Putman, D.; Langer, R., “Accelerated Discovery ofSynthetic Transfection Vectors: Parallel Synthesis and Screening of aDegradable Polymer Library,” J. Am. Chem. Soc. 123, 8155-8156 (2001);Lim, Y. et al., “Biodegradable Polyester,Poly[α-(4-Aminobutyl)-1-Glycolic Acid], as a Non-toxic Gene Carrier,”Pharmaceutical Research 17, 811-816 (2000); Lim, Y., Kim, S., Suh, H. &Park, J.-S., “Biodegradable, Endosome Disruptive, and CationicNetwork-type Polymer as a High Efficient and Non-toxic Gene DeliveryCarrier,” Bioconjugate Chem. 13, 952-957 (2002); Lim, Y. K., S.; Lee,Y.; Lee, W.; Yang, T.; Lee, M.; Suh, H.; Park, J., “CationicHyperbranched Poly(amino ester): A Novel Class of DNA CondensingMolecule with Cationic Surface, Biodegradable Three-DimensionalStructure, and Tertiary Amine Groups in the Interior,” J. Am. Chem. Soc.123, 2460-2461 (2001); and Tuominen, J. et al., “Biodegradation ofLactic Acid Based Polymers under Controlled Composting Conditions andEvaluation of the Ecotoxicological Impact,” Biomacromolecules 3, 445-455(2002). However, under physiological conditions, these cationic polymersare susceptible to degradation via base-catalyzed hydrolysis.

Acid-sensitive polymers containing acetal linkages have been reported,see Tomlinson, R. et al., “Pendent Chain Functionalized Polyacetals ThatDisplay pH-Dependent Degradation: A Platform for the Development ofNovel Polymer Therapeutics,” Macromolecules 35, 473-480 (2002); andMurthy, N., Thng, Y. X., Schuck, S., Xu, M. C. & Fréchet, J. M. J., “ANovel Strategy for Encapsulation and Release of Proteins: Hydrogels andMicrogels with Acid-Labile Acetal Cross-Linkers,” J. Am. Chem. Soc. 124,12398-12399 (2002).

ADDITIONAL REFERENCES

-   Kircheis R, Wightman L, Wagner E. “Design and gene delivery activity    of modified polyethylenimines.” Adv. Drug Deliv. Rev. 2001, 53,    341-358.-   Godbey et al. “Tracking the intracellular path of PEI/DNA complexes    for gene delivery.” Proc. Natl. Acad. Sci. 1999, 96, 5177-5181.-   Chollet et al. “Side-effects of a systemic injection of linear    polyethylenimine-DNA complexes.” J Gene Med 2002, 4, 84-91.-   Tousignant et al. “Comprehensive analysis of the acute toxicities    induced by systemic administration of cationic lipid:plasmid DNA    complexes in mice.” Hum Gene Ther 2000, 11, 2493-2513.-   Escriou et al. “Cationic lipid-mediated gene transfer: effect of    serum on cellular uptake and intracellular fate of lipoamine/DNA    complexes.” Biochim Biophys Acta 1998, 1368, 276-288.-   Plank et al. “Activation of the complement system by synthetic DNA    complexes: a potential barrier for intravenous gene delivery.” Hum    Gene Ther 1996, 7, 1437-1446.-   Ogris et al. “EGylated DNA/transferrin-PEI complexes: reduced    interaction with blood components extended circulation in blood and    potential for systemic gene delivery.” Gene Ther 1999, 6, 595-605.-   Kircheis et al. “Polyethylenimine/DNA complexes shielded by    transferring target gene expression to tumors after systemic    application. Gene Ther 2001, 8, 28-40.-   Trubetskoy et al. “Layer-by-layer deposition of oppositely charged    polyelectrolytes on the Surface of Condensed DNA particles. Nucleic    Acids Res. 1999, 27, 3090-3095.-   Trubetskoy et al. “Recharging cationic DNA complexes with highly    charged polyanions for in vitro and in vivo gene delivery.” Gene    Ther. 2003, 10, 261-271.-   U.S. patent application Ser. No. 10/270,788, filed Oct. 11, 2002    (published as U.S. Patent Publication No. 2003-0215395 A1), and U.S.    Provisional Patent Application Ser. No. 60/378,164, filed May 14,    2002.-   U.S. patent application Ser. No. 10/375,705, filed Feb. 25, 2003    (published as U.S. Patent Publication No. 2004-0166089 A1).-   Tomlinson R, Heller J, Brocchini S, Duncan R.    “Polyacetal-doxorubicin conjugates designed for pH-dependent    degradation.” Bioconjug Chem. 2003, 14(6), 1096-1106.

SUMMARY OF THE INVENTION

It is believed that binary polycation-DNA complexes enter cells by anendocytotic pathway as illustrated in FIG. 6. However, it has been foundthat the ability of such binary complexes to deliver polynucleotides tocells in vivo is limited by toxicity and low gene expression. It isbelieved that decreased transfection efficiency in vivo is caused byinteraction of the positively charged binary polycation-DNA complexeswith negatively charged components such as proteins. The positivecharges of the binary polycation-DNA complex can be shielded bytreatment with polyethyleneglycol (PEG) or proteins such as transferrin,or tertiary complexes may be formed with polycations as shown in FIG. 7.However, in practice it has been found that such additional componentstend to complicate the release and delivery of the DNA.

It has now been found that biodegradable tertiary complexes comprising apolyanion, a polycation and a polynucleotide, as shown in FIG. 8,provide a number of benefits. Thus, a preferred embodiment provides acomplex for delivering a polynucleotide to a cell, comprising apolynucleotide; a polycation; and an acid-degradable polyanion.

Another preferred embodiment provides a method of making such a complex,comprising intermixing a first solution comprising the acid-degradablepolyanion with a second solution comprising the polycation and thepolynucleotide.

Another preferred embodiment provides a method of delivering such acomplex into a cell, comprising contacting the complex with the cell.

These and other embodiments are described in greater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects of the invention will be readily apparent fromthe following description and from the appended drawings, which aremeant to illustrate and not to limit the invention, and wherein:

FIG. 1 shows a reproduction of a photograph showing the results of aretardation assay carried out using complexes formed from an aciddegradable polyanion (polyacetal 5), a biodegradable polycation and DNA.The numbers above the photograph indicate the ratios of polycation toDNA and polyanion to DNA ratio (by weight/weight). The results showthat, for this particular set of conditions, DNA was not released fromthe complex, as compared to a control C (no polymers) and a molecularmarker M. The results show that acid degradable polyanion (polyacetal5), biodegradable polycation and DNA formed a tertiary complex.

FIG. 2 shows a bar graph plotting Relative Light Units (RLU) permilligram of protein for transfection of HepaG2 cells using a tertiarycomplex formed from an acid degradable polyanion (polyacetal 10), abiodegradable polycation, and DNA, as compared to a binary complexformed from the biodegradable polycation and DNA, without the anionicpolymer. The results show that transfection using the tertiary complexwas superior to transfection using the binary complex. The resultsindicate that the tertiary complex is likely to be effective for in vivopolynucleotide delivery. Labeling: Ratio of biodegradable polycation:DNA(by weight/weight) for vertical line bar is 32:1, horizontal line bar is16:1, and grid line bar is 8:1. The numbers on the horizontal axis arethe ratio of polyacetal 10 to DNA (by weight/weight).

FIG. 3 shows a preferred reaction scheme for the preparation of anionicpolyacetals 4-9.

FIG. 4 shows a preferred reaction scheme for the preparation of anionicpolyacetal 10.

FIG. 5 shows a preferred reaction scheme for the preparation ofbiodegradable polycation 12.

FIG. 6 schematically illustrates an endocytoxic pathway for the entry ofa binary polycation-DNA complex into a cell.

FIG. 7 schematically illustrates a tertiary complex of polyanion,polycation and polynucleotide.

FIG. 8 schematically illustrates a biodegradable tertiary complex ofpolyanion, polycation and polynucleotide.

FIG. 9 shows a preferred reaction scheme for the preparation of anionicpolyacetal 13 as described in Example 11.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A preferred embodiment is directed to a tertiary complex comprising apolyanion, a polycation and a polynucleotide, wherein at least one ofthe polyanion and the polycation is biodegradable (e.g.,acid-degradable). Preferably, the polycation is biodegradable and thepolyanion is acid-degradable as illustrated in FIG. 8. Preferably, thepolynucleotide is DNA or RNA. Non-limiting examples of preferredpolynucleotides include plasmid DNA, antisense DNA, DNA oligomer, siRNA,ribozyme, and tetramer.

Polycations are macromolecules with multiple groups that are positivelycharged or capable of being positively charged under physiological oracidic conditions. A quaternary amine is an example of a cationic group;primary, secondary and tertiary amines are examples of groups that arecapable of being positively charged under physiological or acidicconditions. Examples of polycations include polyamine andpoly(ethylenimine). The polycation is typically associated with acounterion such as a negatively charged group on a polyanion, an organicion, or an inorganic ion such as fluoride, chloride, bromide, iodide,nitrate, or sulfate. Preferred biodegradable polycations comprise one ormore degradable recurring units that undergo degradation underphysiological conditions, preferably resulting in scission of thepolycation backbone to form lower molecular weight fragments.Non-limiting examples of preferred degradable recurring units includeester, amide, phosphatediester, acetal, imine, hydrazone, enol, enolether, enamine, ketal, and oxime. Non-limiting examples of preferredbiodegradable polycations thus include polyester-polyamine,polyphosphatediester-polyamine, and polyacetal-polyamine. Biodegradablepolycations may be purchased from commercial sources or prepared bymethods known to those skilled in the art. Preferred polycations arepreferably prepared as described in U.S. patent application Ser. No.10/270,788, filed Oct. 11, 2002 (published as U.S. Patent PublicationNo. 2003-0215395 A1), and U.S. Provisional Patent Application Ser. No.60/378,164, filed May 14, 2002, both of which are hereby incorporated byreference in their entireties and particularly for the purpose ofdescribing the preparation of degradable polycations. The molecularweight of polycations is preferably in the range of about 500 daltons toabout 5,000,000 daltons, more preferably in the range of about 2,000daltons to about 50,000 daltons.

Polyanions are macromolecules with multiple groups that are negativelycharged. Carboxylate (CO₂ ⁻), sulfonate (SO₃ ⁻), and phosphate (PO₃ ⁻)are non-limiting examples of groups that are negatively charged. Thepolyanion is typically associated with a counterion such as a cationicgroup on a polycation, an ammonium ion (NH₄ ⁺), or a metal ion such asLi⁺, Na⁺, K⁺, Rb⁺, Cs⁺, Be²⁺, Mg²⁺, or Ca²⁺. Preferred biodegradablepolyanions comprise one or more degradable recurring units that undergodegradation under physiological conditions, preferably resulting inscission of the polycation backbone to form lower molecular weightfragments. Preferred biodegradable polyanions are acid-degradable.Non-limiting examples of preferred degradable recurring units includeester, amide, phosphatediester, acetal, imine, hydrazone, enol, enolether, enamine, ketal, and oxime. Biodegradable polyanions may bepurchased from commercial sources or prepared by methods known to thoseskilled in the art. Preferred polyanions are polyacetals prepared byhydrolysis of the corresponding polyacetal esters as illustrated in FIG.3. The polyacetal esters are preferably prepared as described in U.S.patent application Ser. No. 10/375,705, filed Feb. 25, 2003 (publishedas U.S. Patent Publication No. 2004-0166089 A1), which is herebyincorporated by reference in its entirety and particularly for thepurpose of describing the preparation of degradable polyacetals. Themolecular weight of polyanions is preferably in the range of about 500to about 5,000,000.

In particularly preferred embodiments, biodegradable polyanions comprisea targeting ligand, preferably a targeting ligand selected from thegroup consisting of galactose, lactose, mannose, peptide, antibody,antibody fragment, and transferrin. Particularly preferred polyanionscomprise recurring units represented by Formula (I):

wherein Y is selected from the group consisting of —(CH₂)₂—,—(CH₂)₂—O—(CH₂)₂—, and —(CH₂)₂—O—(CH₂)₂—O—(CH₂)₂—; wherein W is atargeting ligand selected from the group consisting of galactose,lactose, mannose, transferrin, antibody, antibody fragment, and RGDpeptide; wherein m and n are each individually integers in the range of1 to 100,000; and wherein Z is selected from the group consisting of Li,Na, K, Rb, Cs, Be, Mg, and Ca. For example, a highly preferred polyanioncomprises recurring units represented by formula (II)

wherein Y is selected from the group consisting of —(CH₂)₂—,—(CH₂)₂—O—(CH₂)₂—, and —(CH₂)₂—O—(CH₂)₂—O—(CH₂)₂—; wherein m and n areeach individually integers in the range of one to 100,000; and wherein Zis selected from the group consisting of Li, Na, K, Rb, Cs, Be, Mg, andCa. A polyanion comprising recurring units represented by formula (II)is preferably prepared by hydrolyzing the corresponding polyacetalprecursor to form a hydrolyzed polyacetal; and coupling a galactosaminewith the hydrolyzed polyacetal to form the polyanion. The coupling ispreferably conducted using a coupling reagent selected from the groupconsisting of 1,3-diisopropylcarbodiimide (DIC), N-hydroxysuccinimide(NHS), and 4-dimethylaminopyridine (DMAP). The polyacetal precursor ispreferably prepared as described in U.S. patent application Ser. No.10/375,705, filed Feb. 25, 2003 (published as U.S. Patent PublicationNo. 2004-0166089 A1), which has been incorporated by reference herein asdiscussed above.

Biodegradable tertiary complexes comprising a polyanion, a polycationand a polynucleotide may be prepared in various ways. Preferably, thepolyanion, polycation and polynucleotide are dissolved in solution andintermixed to form a tertiary complex. More preferably, a the polycationand the polynucleotide are intermixed in solution, preferably forming apositively charged binary complex, and the resulting solution is thenpreferably intermixed with the polyanion to form a tertiary complex. Theratios of polycation to polynucleotide and polyanion to polynucleotidemay vary over a broad range. Preferably, the ratio of polycation topolynucleotide is in the range of about 1:1 to about 100:1, morepreferably in the range of about 5:1 to 50:1.

The ratio of polyanion to polynucleotide is preferably selected bytaking into consideration the polycation to polynucleotide ratio, andutilizing an amount of polyanion that is effective to at least partiallyneutralize any excess of cationicity present in a binary complex formedbetween the polycation and polynucleotide. For example, the amount ofpolycation is preferably in excess of the amount of polynucleotide on aweight basis, and thus the amount of cationic charge on the polycationis often in excess of the amount of negative charge on thepolynucleotide. Thus, any binary complex formed by using such amounts ofpolycation and polynucleotide will typically have a net positive charge.Preferably, the amount of polyanion used with such amounts of polycationand polynucleotide is effective to at least partially neutralize the netpositive charge. More preferably, the polyanion is used in an amountthat slightly exceeds the amount effective to neutralize the netpositive charge. Those skilled in the art recognize that the relativeamounts of polycation, polyanion and polynucleotide needed to obtain atertiary complex having the desired nucleotide content and charge levelmay be calculated in advance based on the known or determined chargelevels of the components, and thus formation of a binarypolycation-polynucleotide complex is not required prior to the additionof the polyanion.

In vitro and in vivo delivery of the polynucleotide to the interior of acell (transfection) may be carried out by contacting the tertiarycomplex with the cell to be transfected. Preferably, the cell is amammalian cell. The contacting of the tertiary complex with the cell invitro may be carried out in various ways known to those skilled in theart, e.g., as described in the non-limiting examples set forth below.Contacting of the tertiary complex with the cell in vivo is preferablycarried out by introducing the complex into the body of an animal,preferably a mammal, e.g., by systemic or local administration.Preferably, administration is conducted by identifying a mammal, e.g., ahuman, having cells in need of transfection with a particularpolynucleotide, then administering a tertiary complex comprising thepolynucleotide to the mammal in an amount effective to deliver thedesired amount of polynucleotide to the cells. Such amounts of thetertiary complex effective to deliver desired polynucleotide may bedetermined by routine in vitro experimentation and/or by other methodsknown to those skilled in the art, e.g., clinical trials.

EXAMPLES

Cell lines and cultures used in the following examples were prepared asfollows: Hepatoma (HepaG2) cells were grown in Dulbecco's-modifiedEagle's medium (DMEM) containing 10% (v/v) heat-inactivated fetal bovineserum (FBS), 100 U/ml Penicillin and 100 μg/ml streptomycin, andincubated at 37° C. at 100% humidity atmosphere containing 7.5% CO₂. Theplasmid vector pCMV-luc was constructed by cloning the fireflyluciferase gene into pCMV-0, with the same backbone of mammalianexpression vector. The plasmid was expanded in DH5α E. coli and purifiedwith Qiagen Plasmid Max Preparation Kit according to the manufacture'sinstructions. The quantity and quality of the purified plasmid DNA wasassessed by spectrophotometric analysis at 260 and 280 nm as well as byelectrophoresis in 0.8% agarose gel. Purified plasmid DNA wasresuspended in sterile distilled, deionized H₂O and frozen. Polyacetals1-3 and biodegradable polycation 12 were synthesized in the mannerdescribed in U.S. patent application Ser. No. 10/270,788, filed Oct. 11,2002 (published as U.S. Patent Publication No. 2003-0215395 A1); U.S.Provisional Patent Application Ser. No. 60/378,164, filed May 14, 2002;and U.S. patent application Ser. No. 10/375,705, filed Feb. 25, 2003(published as U.S. Patent Publication No. 2004-0166089 A1). All of thechemicals and reagents for syntheses of polyacetals and biodegradablepolycations were purchased from commercial sources.Poly(ethylenimine)-600 daltons (PEI-600) was purchased fromPolysciences, Inc. Polymer molecular weights were measured by aqueoushigh pressure size exclusion chromatography (HPSEC) using polyethyleneglycol standards. Sizes and zeta potentials of binary and tertiarycomplexes were analyzed by ZetaPALS (zeta potential and particle sizeanalyzer) by following the protocols provided by the manufacturer(Brookhaven Instruments Corporation) Buffers and PEI (branched, 25 kDaltons, Sigma-Aldrich) were obtained commercially.

Examples 1-3

Synthesis of polyacetals 4-6 (FIG. 3): The following description for thesynthesis of anionic polyacetal 5 is illustrative: A solution of lithiumhydroxide monohydrate (0.45 g, 10.7 mmol) in methanol/water (1:1) (20mL) was added to polyacetal 2 (2.57 g, 8.6 mmol). The reaction mixturewas stirred for 24 hours and concentrated by rotary evaporation. Theresidue was redissolved in ethanol (50 mL). Acetone (200 mL) was addedto the resulting solution to form a precipitate. The mixture wasdecanted and the residue was placed under high vacuum to give polyanion5 (1.7 g).

Examples 4-6

Synthesis of polyacetals 7-9 (FIG. 3): The following description for thesynthesis of polyacetal 8 is illustrative: A solution of1,3-diisopropylcarbodiimide (DIC, 0.082 g, 0.65 mmol) in DMF (10 mL) wasadded to anionic polyacetal 5 (0.39 g, 1.3 mmol). N-hydroxysuccinimide(NHS, 0.075 g, 0.65 mmol) in DMF (15 mL) was added into the reactionmixture. The reaction was stirred for 7 hours. The precipitate wasisolated and washed with more DMF and placed under high vacuum to give 8(0.45 g).

Example 7

Synthesis of polyacetal 10: A solution of galactosamine-HCl (0.11 g,0.51 mmol) and 4-dimethylaminopyridine (DMAP, 0.12 g, 0.98 mmol) indimethylsulfoxide (DMSO, 2 mL) was added to a solution of polyanion 8(0.20 g) in PBS (8 mL). The reaction mixture was stirred for 15 hours.The reaction solution was added with acetone (200 mL) and stirred.Within 1 hour, precipitate formed. The precipitate was redissolved indistilled water (2 mL) and dialyzed in distilled water (2000 mL) for 5hours. The solution was placed under high vacuum to give 10.

Example 8

The polycation 12 used in the following examples was prepared asdescribed in U.S. Ser. No. 10/270,788, filed Oct. 11, 2002 (published asU.S. Patent Publication No. 2003-0215395 A1), and U.S. 60/378,164, filedMay 14, 2002, as illustrated in FIG. 5.

Example 9

Tertiary complexation of polyacetal 5 to form DNA/polycation complexes:Samples of plasmid DNA and biodegradable polycation 12 were diluted inOptimem solution (Life Technologies) and mixed to form a series ofbinary polycation/DNA complexes having polycation to DNA ratios rangingfrom 32:1 to 8:1 by weight. After about 15 minutes, a solution ofpolyanion 5 in Optimem solution was added into the polycation/DNAcomplexes and incubated at room temperature for another 15 minutes. Theresulting tertiary complexes were added to agarose gel andelectrophoresis was performed: Five μl of loading dye was added to eachsample, and 15 μl of each sample were loaded per well. The tertiarycomplexes were analyzed by electrophoresis in a 0.3% agarose gel with0.04 M Tris-acetate buffer, pH 7.4, containing 1 mM EDTA, at 100V for 30minutes. The tertiary complexes were visualized by UV illumination. Theretardation assay results for the tertiary polycation/polyanion/DNAcomplexes are shown in FIG. 1. The results indicate that the tertiarycomplexes were successfully formed and that polyanion 5 did not competewith DNA, so that DNA was released from the tertiary complexes at eachof the ratios.

Example 10

In vitro transfection using a tertiary complex comprising polycation 12and anionic polyacetal 10 was carried out as follows: HepaG2 cells wereplated in 24-well tissue culture plates (2×10⁵ cells/well for HepaG2cells) and incubated overnight in DMEM with 10% fetal bovine serum(FBS). For each well, a 20 μl aliquot of polycation 12 solution (eachcontaining a different dose of biodegradable polycation 12) was addeddropwise into a 20-μl DNA solution containing 0.6 μg of plasmid DNA(pCMV-GFP plasmid DNA or pCMV-luc), while vortexing. Dropwise additionwhile vortexing was found to be highly preferable, because it was foundthat transfection results depended on the mixing conditions. Thesolutions containing DNA and polycation 12 were incubated for 15 min atroom temperature to form binary DNA-polycation 12 complexes. Then 20 mlof solutions containing anionic polyacetal 10 were added to each of thebinary DNA-polycation 12 complexes to form tertiary complexes(containing DNA, anionic polyacetal 10, and polycation 12). Next, 60 mLsamples of these tertiary complexes were added into each well and thecells were incubated (37° C., 7.5% CO₂) for 24 hours. After thatincubation, fruitfly luciferase activities were detected as describedbelow.

Luciferase activity was measured using a chemiluminescent assayfollowing the manufacturer's instructions (Luciferase Assay System,Promega, Madison, Wis., USA). About twenty four hours after thetransfections described above, the cells were rinsed twice with PBS andthen were lysed with lysis buffer (1% Triton X-100, 100 mM K₃PO₄, 2 mMdithiothreitol, 10% glycerol, and 2 mM EDTA pH 7.8) for 15 min at roomtemperature. A 10-μl aliquot of cell lysate was then mixed with 50-μl ofluciferase assay reagent with the injector at room temperature in theluminometer. Light emission was measured in triplicate over 10 secondsand expressed as RLUs (relative light units). Relative light units (RLU)were normalized to the protein content of each sample, determined by BSAprotein assay (Pierce, Rockford, Ill.). All the experiments wereconducted in triplicate. The results obtained for the transfection ofHepaG2 cells with pCMV-luc using the tertiary complexes (containing DNA,anionic polyacetal 10, and polycation 12) are shown in FIG. 2. Theresults show that tertiary complexes containing polyacetal 10 providedincreased transfection efficiency. The results also indicated that thetertiary complexes would be effective for in vivo polynucleotidedelivery.

Example 11

Synthesis of polyacetal 13 (FIG. 9): 1,2,4,5-Benzenetetracarboxylicdianhydride (5.0 g, 22.9 mmol, Aldrich Chemical Company) was added to asolution of 3,9-bis(3-aminopropyl)-2,4,8,10-tetraoxaspiro[5.5]undecane(6.34 g, 22.9 mmol TCI Chemical Company) in dimethylacetamide (DMA, 100mL,). The mixture was stirred for 3 days at room temperature. Thereaction was quenched with saturated sodium bicarbonate carbonate inwater (50 mL). The precipitate was filtered, and the filtrate was pouredin acetone (1000 mL) and white precipitate formed. The precipitate wasredissolved in distilled water and dialyzed in cellulose membrane (2,000molecular weight cut-off) in distilled water for 15 hours with 4 timesof water changing. Clear solid polyacetal 13 (3.2 g) was obtained afterthe water was removed by rotary evaporation. The molecular weight of thepolyacetal 13 was about 45,000 daltons (by HPSEC, polyethylene glycolstandards). ¹H- and ¹³C NMR spectra of polyacetal 13 were obtained andfound to be consistent with the chemical structure of polyacetal 13shown in FIG. 9. In FIG. 9, n and m are each individually integers inthe range of about 1 to about 200. Control over the molecular weight ofthe polyacetal 13 (and thus over m and n) may be exercised by varyingthe molar ratio of the dianhydride monomer to the diamine monomer.

Example 12

Sizes and zeta potentials of binary polycation PEI-DNA and biodegradablepolycation 12-DNA complexes were determined as follows:

Size Measurements: A series of solutions containing different amounts ofpolycation PEI or biodegradable polycation 12 (50 μL) in HEPES bufferswas added to solutions containing DNA (2 μg, 50 μL) in HEPES buffers bypipetting, agitated by pipetting, then allowed to equilibrate for 1minute. Particle sizes (nm) of the resulting binary complexes weremeasured by ZetaPALS, and the results are summarized in Table 1 below.

Zeta potential measurements: Selected different amounts of PEI orpolycation 12 were added to a solution of DNA with similarconcentrations as in the size measurements but scaled-up to 1.6 mL totalvolume. Zeta potentials (mV) of the resulting binary complexes weremeasured by ZetaPALS, and the results are summarized in Table 1 below.

TABLE 1 Amount of PEI (μg) 0.125 0.25 0.5 1 2 4 8 16 32 64 128 256 512PEI:DNA 0.06 0.1 0.25 0.5 1 2 4 8 16 32 64 128 256 ratio (w:w) Particle124.1 103 153 112 94.6 78.6 82.5 88.1 80.4 69 55.8 63.6 91 size (nm)Zeta −29 49 51 potential (mV) Poly- 0.311 0.21 0.11 0.2 0.21 0.23 0.210.27 0.25 0.2 0.33 0.3 0.3 dispersity Avg. Count 23.9 73.4 497 335 203143 158 167 161 151 169 200 305 Rate (kcps) Baseline 0 2.7 7.8 6.5 109.7 8.6 0 0 9.8 8.2 7.2 9.9 Index Amount of biodegradable polycation 12(μg) 0.25 0.5 1 2 4 8 16 32 64 128 256 12:DNA 0.1 0.25 0.5 1 2 4 8 16 3264 128 ratio (w:w) Particle 200 106 110 1316 91.2 88.7 80.4 77.3 74 72.871.3 size (nm) Zeta −27 19 38.2 potential (mV) Poly- 0.3 0.21 0.1 0.30.12 0.13 0.19 0.19 0.2 0.2 0.2 dispersity Avg. Count 18.1 61.9 227 277274 259 162 143 126 121 118 Rate (kcps) Baseline 0 5.1 8.3 9.1 8.9 8.97.8 9.3 9.4 9.8 8.3 Index

Example 13

Sizes and zeta potentials of tertiary complexes were determined asfollows:

Size Measurements: A series of solutions containing different amounts ofpolyacetal 13 (100 μL) in buffers were added to solutions containingbinary PEI and DNA complexes (8 μg and 2 μg, respectively, 100 mL) inHEPES buffers by pipetting, agitated by pipetting, then allowed toequilibrate for 1 minute. Particle sizes (nm) of the resulting ternarycomplexes were measured by ZetaPals, and the results are summarized inTable 2 below.

Zeta potential measurements: A series of solutions containing differentamounts of polyacetal 13 in buffers were added to solutions of binaryPEI and DNA complexes with similar concentrations as in the sizemeasurements but scaled-up to 1.6 mL total volume. Zeta potentials (mV)of the resulting ternary complexes were measured by ZetaPals, and theresults are summarized in Table 2 below.

TABLE 2 Amount of Polyacetal 13 (μg) (HEPES buffer) 500 250 125 64 32 1613:PEI:DNA 500:8:2 250:8:2 125:8:2 64:8:2 32:8:2 16:8:2 (w/w)measurement 177 146 138 140 Turbidity 121 1 (nm) measurement 182 145 137143 Turbidity 99 2 (nm) measurement 173 154 136 143 Turbidity 114 3 (nm)zeta (mV) −37 −51 −28 −25 42 Amount of Polyacetal 13 (μg) (BIGS buffer)¹500 250 125 64 32 16 13:PEI:DNA 500:8:2 250:8:2 125:8:2 64:8:2 32:8:216:8:2 (w/w) measurement 183.6 170.1 148.1 150.6 Turbidity 157.8 1 (nm)measurement 185.2 153.0 157.5 143.0 Turbidity 131.2 2 (nm) measurement178.4 161.1 154.0 166.5 Turbidity 142.2 3 (nm) zeta (mV) −34 −30 −25 −32−12 38 ¹BIGS buffers are 10% HEPES, 5% glucose, pH 7.4

Example 14

Safety studies of the polyacetal 13: 15 hairless mice (SKH1 model, 5-6weeks old, 18-20 grams) were purchased from Charles River Laboratoriesand were kept at the animal facility for 3 days before conductingexperiments. 15 mice were divided into 3 groups (5 mice per group).Polyacetal 13 samples were prepared by dissolving the polymer in PBS (pH7.4) at three different concentrations: (1) 40 mg/mL, (2) 20 mg/mL, and(3) 10 mg/mL). The mice were then injected (tail vein) with 100 μL ofeach concentration (one injection per mouse). The mice were observed for5 hours and results are shown in Table 3 below:

TABLE 3 Polyacetal 13 Dose (μg) 1,000 2,000 4,000 # of animals injected5 5 5 # of animals alive 5 5 5 Behavior Active Active Active

It will be appreciated by those skilled in the art that variousomissions, additions and modifications may be made to the processes andcompositions described above without departing from the scope of theinvention, and all such modifications and changes are intended to fallwithin the scope of the invention, as defined by the appended claims.

1. A polyanion comprising recurring units represented by Formula (I):

wherein Y is selected from the group consisting of —(CH₂)₂—,—(CH₂)₂—O—(CH₂)₂—, and —(CH₂)₂—O—(CH₂)₂—O—(CH₂)₂—; wherein W is atargeting ligand selected from the group consisting of galactose,lactose, mannose, transferrin, antibody, antibody fragment, and RGDpeptide; wherein m and n are each individually integers in the range of1 to 100,000; and wherein Z is selected from the group consisting of Li,Na, K, Rb, Cs, Be, Mg, and Ca.
 2. The polyanion of claim 1, wherein Y is—(CH₂)₂—O—(CH₂)₂—O—(CH₂)₂—.
 3. The polyanion of claim 1, wherein Y is—(CH₂)₂—O—(CH₂)₂—.
 4. The polyanion of claim 1, wherein Y is —(CH₂)₂—.5. The polyanion of claim 1, wherein Z is Li or Na.
 6. The polyanion ofclaim 1, wherein the polyanion is acid degradable.
 7. The polyanion ofclaim 1, wherein the polyanion has a molecular weight in the range ofabout 500 Daltons to about 5,000,000 Daltons.
 8. The polyanion of claim1, comprising recurring units represented by formula (II):

wherein Y is selected from the group consisting of —(CH₂)₂—,—(CH₂)₂—O—(CH₂)₂—, and —(CH₂)₂—O—(CH₂)₂—O—(CH₂)₂—; wherein m and n areeach individually integers in the range of 1 to 100,000; and wherein Zis selected from the group consisting of Li, Na, K, Rb, Cs, Be, Mg, andCa.
 9. The polyanion of claim 8, wherein Z is Li or Na.
 10. A method formaking the polyanion of claim 1, comprising: hydrolyzing a polyacetal toform a hydrolyzed polyacetal; and coupling a galactosamine with thehydrolyzed polyacetal to form the polyanion of claim
 1. 11. The methodof claim 10, wherein the polyacetal comprises a recurring unit havingthe following structure:

wherein Y is selected from the group consisting of —(CH₂)₂—,—(CH₂)₂—O—(CH₂)₂—, and —(CH₂)₂—O—(CH₂)₂—O—(CH₂)₂—; and wherein m is aninteger in the range of 1 to 100,000.
 12. The method of claim 11,wherein Y is —(CH₂)₂—O—(CH₂)₂—.
 13. The method of claim 10, wherein thehydrolyzed polyacetal comprises a recurring unit having the followingstructure:

wherein Y is selected from the group consisting of —(CH₂)₂—,—(CH₂)₂—O—(CH₂)₂—, and —(CH₂)₂—O—(CH₂)₂—O—(CH₂)₂—; wherein m is aninteger in the range of 1 to 100,000; and wherein Z is selected from thegroup consisting of Li, Na, K, Rb, Cs, Be, Mg, and Ca.
 14. The method ofclaim 13, wherein Z is Li or Na.
 15. The method of claim 13, wherein Yis —(CH₂)₂—O—(CH₂)₂—.
 16. The method of claim 10 in which the couplingis conducted using a coupling reagent selected from the group consistingof DIC, NHS and DMAP.
 17. The method of claim 10 in which the couplingis conducted using DMAP.
 18. A polyanion comprising recurring unitsrepresented by Formula (III):

wherein n and m are each individually integers in the range of about 1to about
 200. 19. The polyanion of claim 18, wherein the polyanion ofFormula (III) has a molecular weight in the range of about 500 Daltonsto about 5,000,000 Daltons.
 20. The polyanion of claim 19, wherein thepolyanion of Formula (III) has a molecular weight of about 45,000Daltons.